Tsing B. Chen

Two angiotensin II binding sites in rat brain revealed using [125I]Sar1, Ile8-angiotensin II and selective nonpeptide antagonists

[125I]Sar1, Ile8 angiotensin II labeled two distinct binding sites in rat brain. The displacement potencies of WL-19, a selective ligand for the angiotensin II subtype 2 receptor, angiotensin II and related peptides indicated that one binding site in the rat brain is the same as the adrenal angiotensin subtype 2 receptor. The second binding site in rat brain was displaced by the selective angiotensin II subtype 1 receptor antagonist DuP-753; however, the displacement potencies of angiotensin II, angiotensin III and Ile7-angiotensin III were significantly less than at the adrenal angiotensin subtype 1 receptor. The data suggests that this binding site in rat brain may represent an angiotensin II receptor subtype which shares some characteristics with the adrenal angiotensin subtype 1 receptor.

Increase in brain 125I-cholecystokinin (CCK) receptor binding following chronic haloperidol treatment, intracisternal 6-hydroxydopamine or ventral tegmental lesions

Specific 125I-CCK receptor binding was significantly increased in brain tissue taken from guinea pig or mouse following chronic (2-3 week) daily administration of haloperidol (2-3 mg/kg/day). Scatchard analysis indicated the increase in CCK binding was due to an increased receptor number (B max) with no change in affinity (Kd). In guinea pigs, the increased CCK binding was observed in the mesolimbic regions and frontal cortex, but not in striatum, hippocampus nor posterior cortex. In mice, however, the increases occurred in both pooled cerebral cortical-hippocampal tissue, and in the remainder of the brain. Enhanced CCK receptor binding was also observed in membranes prepared from whole brain of mice one month following intracisternal injection of 6-hydroxydopamine. Additionally, an increase in CCK binding was observed in mesolimbic regions and frontal cortex, but not striatum or hippocampus, of guinea pigs 3 weeks after an unilateral radiofrequency lesions of the ipsilateral ventral tegmentum. The present studies demonstrate that three different procedures which reduce dopaminergic function in the brain enhance CCK receptor binding. The data provide further support for a functional interrelationship between dopaminergic systems and CCK in some brain regions and raise the possibility that CCK may play a role in the antipsychotic action of neuroleptics.

Tifluadom, a κ-opiate agonist, acts as a peripheral cholecystokinin receptor antagonist

Abstract Tifluadom, a benzodiazepine κ -opiate agonist, stereoselectively inhibited the binding of 125 I-CCK to pancreatic membranes (IC 50 =47 nM). Several other opiate agonists were ineffective. Scatchard analysis indicated the inhibition of CCK binding by tifluadom was competitive in nature. Tifluadom (1 μM) did not displace 125 I-CCK binding to brain tissue or 125 I-gastrin binding to fundic glands. In the isolated guinea pig gallbladder, tifluadom antagonized CCK-8 induced contractions with an estimated pA 2 of 6.8. These data demonstrate that tifluadom is a peripherally selective CCK antagonist. This unique action could contribute to its reported analgesic and appetite stimulatory properties.

Design and synthesis of N-alkylated saccharins as selective α-1a adrenergic receptor antagonists☆

Abstract Benign prostatic hyperplasia can be managed pharmacologically with α-1 adrenergic receptor antagonists. Agents that demonstrate selectivity for the α-1 a receptor subtype may offer advantages in clinical applications with respect to hypotensive side effects. The N-alkylated saccharins reported here represent a new class of subtype selective α-1 a adrenergic receptor antagonists which demonstrate potent effects on prostate function in vivo and are devoid of blood pressure side effects.

Design and Synthesis of Novel α1a Adrenoceptor-Selective Antagonists. 3. Approaches To Eliminate Opioid Agonist Metabolites by Using Substituted Phenylpiperazine Side Chains

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.

Characterization of [3H](±)L-364,718 binding to solubilized cholecystokinin (CCK) receptors of rat pancreas

The binding of [3H](+/-)L-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. [3H](+/-)L-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound [3H](+/-)L-364,718. Specific [3H](+/-)L-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific [3H](+/-)L-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific [3H](+/-)L-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that [3H](+/-)L-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by [3H](+/-)L-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.

Non-invasive bioluminescence imaging of β-cell function in obese-hyperglycemic [ob/ob] mice.

Background Type 2 diabetes results from failure of the β-cells to compensate for increased insulin demand due to abnormal levels of metabolic factors. The ob/ob(lep-/-) mouse has been extensively studied as an animal model of type 2 diabetes. Previous studies have shown a correlation between β-cell function and bioluminescent imaging in lean genetically engineered mice. The ability to noninvasively monitor β-cell function in ob/ob mice could provide new information on β-cell regulation in type 2 diabetes. Methods To create the B6 Albino ob/ob MIP-luc mice (ob/ob-luc), the ob/ob mouse was crossed with the CD1 MIP-luc mouse. All mice were backcrossed over multiple generations to ensure the genetic background of the transgenic mice was over 96% similar to the background of the original ob/ob mouse. Animal weight, blood glucose levels, insulin in plasma, and in vivo bioluminescence (BLI) were monitored weekly or biweekly for up to 70 weeks of age. BL imaging was performed using IVIS Spectrum (Perkin Elmer) and calculated by integrating the bioluminescence signal between 5 and 10 min after i.v. injection of D-luciferin. Insulin immunohistochemistry determined islet beta cell count and insulin secretion assay determined islet insulin function. Results There were significant increases in BLI and insulin levels as the ob/ob-luc mice aged while glucose levels gradually decreased. Ob/ob-luc were sacrificed at different time points to determine ex vivo BLI, islet function and total β-cell numbers using a cell counting training algorithm developed for the Vectra image analysis system (Perkin Elmer). The number of β-cells increased as the mice aged and all three ex vivo measurements correlated with BLI. Conclusions The ob/ob-luc mice can serve as a model of metabolic stress, similar to human type 2 diabetes using BLI as a surrogate marker for β-cell function.

Design and synthesis of novel dihydropyridine alpha-1A antagonists

A series of analogs of SNAP 5150 containing heteroatoms at C2 or C6 positions is described. Herein, we report that the presence of alkyl substituted heteroatoms at the C2(6)-positions of the dihydropyridine are well tolerated. In addition, 15 inhibited the phenylephrine induced contraction of dog prostate tissue with a Kb of 1.5 nM and showed a Kb (DBP, dogs, microg/kg)/Kb (IUP, dogs, microg/kg) ratio of 14.8/2.5.